*Background/Disease Information(sort of like the Intro to your Mini Research Write up): Plasmodium falciparum is a protozoan parasite that causes the most dangerous strain of malaria in humans. In 2012, malaria caused about 627,000 deaths mostly among African children. This disease is transmitted only through Anopheles mosquito bites and is most prevalent in tropical areas where the mosquitoes can breed in shallow water. Malaria is curable and preventable and recent studies have shown that the enoyl reductase enzyme (ENR) that is responsible for type II fatty-acid biosynthesis pathway in the protozoa has been inhibited by the antibacterial agent Triclosan.
Complex of proteins?: in complex with NADH Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below) http://www.sigmaaldrich.com/catalog/product/sigma/28007?lang=en®ion=US
-- links to assay reagents (substrates) pages. http://www.brenda-enzymes.info/literature/lit.php4?e=1.3.1.9&r=390898
--- List cost and quantity of substrate reagents, supplier, and catalog # Substrate reagent: Crotonoyl coenzyme A trilithium salt Cost: 5mg = $266.50 ; 25mg = $920.00 Supplier: Sigma-Aldrich Catalog #: MFCD00151226
Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 1NHW
Current Inhibitors: 5-chloro-2-(2,4-dichlorophenoxy)phenol Expression Information (has it been expressed in bacterial cells): Although the TDR page showed no information on expression, BRENDA shows that this protein has been expressed in Escherichia coli
Purification Method:
There are two different vectors that can be used to purify the protein: pMALc2x and pMALcHT vectors. pMALc2x:
Cells were resuspended in lysis buffer (20mM sodium/potassium phosphate pH 7.5, 1 mg/mL lysozyme, 2.5 micrograms/mL DNAse I, 200 mM NaCl), centrifuged, and applied to a 10mL amylose column for affinity purification. Purified MBP-ENR fusion protein was digested with factor Xa and desalted with a HiPrep 26/10 desalting column and applied to an SP Sepharose cation-exchange column. pMALcHT:
Cells were clarified by centrifugation and applied to a 5 mL HiTrap Chelating HP colum and desalted with a HiPrep 26/10 desalting column and loaded onto a 5mL HiTrap SP Fast Flow column.
Source: http://journals.iucr.org/d/issues/2003/07/00/za0101/za0101.pdf
Image of protein (PyMol with features delineated and shown separately):
Figure 1: 1NHW enoyl-acyl-carrier-protein reductase viewed in PyMol, shown as cartoon with ligands shown in sticks.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MNKISQRLLFLFLHFYTIVCFIQNNTQKTFHNVLQNEQIRGKEKAFYRKEKRENIFIGNK MKHLNNMNNTHNNNHYMEKEEQDASNINKIKEENKNEDICFIAGIGDTNGYGWGIAKELSKRNVKIIFGIWPPVYNIFMKNYKNGKFDNDMIIDKDKKMNILDMLPFDASFDTANDIDEETKNNKRYNMLQNYTIEDVANLIHQKYGKINMLVHSLANAKEVQKDLLNTSRKGYLDALSKSSYSLISLCKYFVNIMKPQSSIISLTYHASQKVVPGYGGGMSSAKAALESDTRVLAYHLGRNYNIRINTISAGPLKSRAATAINKLNNTYENNTNQNKNRNSHDVHNIMNNSGEKEEKKNSASQNYTFIDYAIEYSEKYAPLRQKLLSTDIGSVASFLLSRESRAITGQTIYVDNGLNIMFLPDDIYRNENE
*length of your protein in Amino Acids: 432 amino acids Molecular Weightof your protein in kiloDaltons using theExpasy ProtParamwebsite 49.7623 kDA MolarExtinction coefficientof your protein at 280 nm wavelength: Ext. coefficient 45395 Abs 0.1% (=1 g/l) 0.912, assuming all pairs of Cys residues form cystines
Ext. coefficient 45270 Abs 0.1% (=1 g/l) 0.910, assuming all Cys residues are reduced
*GC% Content for gene:28.098537336413% *CDS Gene Sequence (codon optimized) - copy fromoutputofPrimer DesignProtocol (paste as text only): N/A *GC% Content for gene (codon optimized): N/A
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works outputtext file-that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#:46361102
*Protein ID (NP or XP #) or Wolbachia#:XP_966137.1
*Organism (including strain): Plasmodium falciparum 3D7
Etiologic Risk Group (see link below): Risk group level 3
http://www.absa.org/riskgroups/parasitessearch.php?genus=plasmodium&species=falciparum
*Background/Disease Information(sort of like the Intro to your Mini Research Write up):
Plasmodium falciparum is a protozoan parasite that causes the most dangerous strain of malaria in humans. In 2012, malaria caused about 627,000 deaths mostly among African children. This disease is transmitted only through Anopheles mosquito bites and is most prevalent in tropical areas where the mosquitoes can breed in shallow water. Malaria is curable and preventable and recent studies have shown that the enoyl reductase enzyme (ENR) that is responsible for type II fatty-acid biosynthesis pathway in the protozoa has been inhibited by the antibacterial agent Triclosan.
Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=4502
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://plasmodb.org/plasmo/showRecord.doname=GeneRecordClasses.GeneRecordClass&primary_key=PFF0730c
Essentiality of this protein:
PFF0730c has essentiality data
Gene/Ortholog: mtu1508 (OG4_13747); Phenotype: non-essential; Source study: nmpdr
Gene/Ortholog: eco1251 (OG4_13747); Phenotype: undefined; Source study: blattner
Gene/Ortholog: eco1251 (OG4_13747); Phenotype: essential; Source study: gerdes
Gene/Ortholog: eco1251 (OG4_13747); Phenotype: essential; Source study: keio
Gene/Ortholog: eco1251 (OG4_13747); Phenotype: essential; Source study: shigen
The enoyl-acyl carrier protein reductase catalyzes the final step in fatty acid elongation type II pathway in Plasmodium falciparum.
Source: http://www.jbc.org/content/277/15/13106.full.pdf+html
Complex of proteins?: in complex with NADH
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8
*EC#: 1.3.1.9
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.3.1.9&Suchword=&organism%5B%5D=Plasmodium+falciparum&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below) http://www.sigmaaldrich.com/catalog/product/sigma/28007?lang=en®ion=US
-- links to assay reagents (substrates) pages. http://www.brenda-enzymes.info/literature/lit.php4?e=1.3.1.9&r=390898
--- List cost and quantity of substrate reagents, supplier, and catalog #
Substrate reagent: Crotonoyl coenzyme A trilithium salt
Cost: 5mg = $266.50 ; 25mg = $920.00
Supplier: Sigma-Aldrich
Catalog #: MFCD00151226
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1NHW
Current Inhibitors: 5-chloro-2-(2,4-dichlorophenoxy)phenol
Expression Information (has it been expressed in bacterial cells):
Although the TDR page showed no information on expression, BRENDA shows that this protein has been expressed in Escherichia coli
Purification Method:
There are two different vectors that can be used to purify the protein: pMALc2x and pMALcHT vectors.
pMALc2x:
Cells were resuspended in lysis buffer (20mM sodium/potassium phosphate pH 7.5, 1 mg/mL lysozyme, 2.5 micrograms/mL DNAse I, 200 mM NaCl), centrifuged, and applied to a 10mL amylose column for affinity purification. Purified MBP-ENR fusion protein was digested with factor Xa and desalted with a HiPrep 26/10 desalting column and applied to an SP Sepharose cation-exchange column.
pMALcHT:
Cells were clarified by centrifugation and applied to a 5 mL HiTrap Chelating HP colum and desalted with a HiPrep 26/10 desalting column and loaded onto a 5mL HiTrap SP Fast Flow column.
Source: http://journals.iucr.org/d/issues/2003/07/00/za0101/za0101.pdf
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MNKISQRLLFLFLHFYTIVCFIQNNTQKTFHNVLQNEQIRGKEKAFYRKEKRENIFIGNK
MKHLNNMNNTHNNNHYMEKEEQDASNINKIKEENKNEDICFIAGIGDTNGYGWGIAKELSKRNVKIIFGIWPPVYNIFMKNYKNGKFDNDMIIDKDKKMNILDMLPFDASFDTANDIDEETKNNKRYNMLQNYTIEDVANLIHQKYGKINMLVHSLANAKEVQKDLLNTSRKGYLDALSKSSYSLISLCKYFVNIMKPQSSIISLTYHASQKVVPGYGGGMSSAKAALESDTRVLAYHLGRNYNIRINTISAGPLKSRAATAINKLNNTYENNTNQNKNRNSHDVHNIMNNSGEKEEKKNSASQNYTFIDYAIEYSEKYAPLRQKLLSTDIGSVASFLLSRESRAITGQTIYVDNGLNIMFLPDDIYRNENE
*length of your protein in Amino Acids: 432 amino acids
Molecular Weightof your protein in kiloDaltons using theExpasy ProtParamwebsite 49.7623 kDA
MolarExtinction coefficientof your protein at 280 nm wavelength:
Ext. coefficient 45395
Abs 0.1% (=1 g/l) 0.912, assuming all pairs of Cys residues form cystines
Ext. coefficient 45270
Abs 0.1% (=1 g/l) 0.910, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html)
*CDS gene sequence (paste as text only):
1 atgaataaaa tatcacaacg gttattattc ctctttctac atttttatac cattgtgtgc
61 tttatccaga ataatactca aaagacattc cataacgttc ttcagaatga acagatacga
121 ggtaaggaaa aagcatttta taggaaagaa aaacgcgaaa acatatttat aggaaacaaa
181 atgaaacact taaataatat gaataataca cacaataata atcattatat ggagaaagaa
241 gaacaagatg catcaaacat aaacaaaatt aaagaagaaa ataaaaatga agatatttgt
301 tttattgctg gtataggaga tacaaatgga tacgggtggg gtattgctaa agaattatcc
361 aaaagaaatg taaaaataat tttcggtatt tggcctcctg tttataatat ttttatgaaa
421 aattataaaa acgggaaatt tgataatgat atgattattg ataaagataa gaaaatgaat
481 atattagaca tgctaccctt tgacgcttct tttgatactg ccaatgatat agatgaagaa
541 acgaaaaata ataaaagata taatatgtta cagaattata ctatagaaga tgttgccaat
601 ttaatacatc aaaaatatgg aaaaattaat atgctcgttc attctttagc taacgctaaa
661 gaagttcaaa aagatttatt aaataccagt cgaaaaggtt atttagatgc tcttagtaaa
721 agttcttact ctttaatatc attatgtaaa tattttgtca atattatgaa acctcaatcc
781 agcattattt cattaacata tcatgctagc caaaaagttg tgccaggcta tggtggaggt
841 atgtccagcg ctaaagctgc actcgaatct gataccagag ttttagctta tcatctagga
901 agaaattata atatcagaat taatactatc agtgcaggac cacttaaatc aagagcagct
961 actgctatta ataaattaaa caatacatat gaaaataata caaatcaaaa taaaaatagg
1021 aatagtcatg atgttcataa tattatgaac aattcaggtg aaaaggaaga aaaaaaaaat
1081 tcagctagcc aaaattatac atttatagat tatgcaatag agtattcaga aaagtatgct
1141 ccattacgtc aaaagttatt atcaacagat attggatctg tagcatcatt tttgttatct
1201 cgagagagtc gtgcgataac tgggcagaca atatatgtgg ataacggatt aaatataatg
1261 tttttaccag atgatatata tcgcaatgaa aatgaataa
*GC% Content for gene: 28.098537336413%
*CDS Gene Sequence (codon optimized) - copy fromoutputofPrimer DesignProtocol (paste as text only): N/A
*GC% Content for gene (codon optimized): N/A
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works outputtext file-that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
code